Physical Characteristics of a Citrullinated Pro-Filaggrin Epitope Recognized by Anti-Citrullinated Protein Antibodies in Rheumatoid Arthritis Sera

Abstract

Rheumatoid arthritis (RA) is an autoimmune disease of complex etiology. A characteristic feature of a subset of RA is the presence of anti-citrullinated protein antibodies (ACPA), which correlate with a progressive disease course. In this study, we employed streptavidin capture enzyme-linked immunosorbent assay to analyze ACPA reactivity. Using the pro-filaggrin peptide HQCHQEST-Cit-GRSRGRCGRSGS, as template, we analyzed the reactivity of RA sera and healthy donor sera to various peptides in order to determine the physical characteristics of the citrullinated pro-filaggrin epitope and to examine whether biotin labelling influence antibody recognition. The full-length cyclic pro-filaggrin peptide and a linear form with a N-terminal biotin, was recognized to the same level, whereas, a notable difference in ACPA reactivity to the linear peptides with a C-terminal biotin was found, probably due to steric hindrance. Screening of linear and cyclic truncated peptides, revealed that small cyclic peptides containing 10–12 amino acids are favored over the linear. Moreover, the charged amino acids C-terminal to citrulline were found to be essential for antibody reactivity, most important was the charged amino acid in position 4 C-terminal to citrulline. Collectively, peptide structure, length, the presence of charged amino acids and biotin labelling markedly influence antibody reactivity. In relation to the clinical diagnostics of ACPA, these findings may reflect the differences in diagnostic assays used for detection of ACPA, which relates to differences in sensitivity and specificity dependent on the assay applied.

Seropositivity and Antibody Profiling of Patients Are Dramatically Impacted by the Features of Peptides Used as Immunosorbents: A Lesson from Anti–Citrullinated Protein/Peptide Antibody

“…Our data not only sustain conclusions obtained by others with citrullinated or noncitrullinated Ags (12,34,39,40) but also raised the question of which peptide characteristic had a major impact in the ELISA results. Indeed, only a few works have investigated the influence of the biotin position or the epitope-biotin distance, on single epitope recognition (11,34,40). With various peptides encompassing a single epitope in different conformations we showed that biotin-epitope distance was the major significant parameter that influenced epitope recognition.…”

Antibodies to a strain-specific citrullinated Epstein-Barr virus peptide diagnoses rheumatoid arthritis

“…Peptide 17, yielding the highest sensitivity, was only present in the EBV AG876 strain, whereas peptides 7 (P = 0.0020) and 8 (P = 0.0010) from the EBV GD1 and EBV B95-8 strains, respectively, yielded sensitivities of 60%. As seen, all three peptides contain repetitive Arg-Gly motifs, which previously have been reported for ACPA epitopes 8 , 10 , 27 , 28 , 30 .…”

“…Highly significant reactivity was found to peptide 17 (P < 0.0001), which yielded a sensitivity of 93%. Interestingly, the two peptides, which were not recognised by the RA sera, contained no charged amino acids C-terminal to Cit, which previously has been reported to be essential for ACPA reactivity 13 , 28 , 29 . None of the HD sera reacted with the citrullinated peptides, which is illustrated by specificities of 100%.…”

Structural Basis of Cross‐Reactivity of Anti–Citrullinated Protein Antibodies

“…By comparing the conformation of the 3 peptides bound to E4 in the crystals with their native forms (i.e., CII and α‐enolase), we found that they indeed undergo substantial conformational rearrangements upon binding to E4. The fact that the peptide conformation is essential for autoantibody reactivity is a finding that has been observed previously .…”